Abstract Our TSRI-ARC has focused on preclinical work on the cellular, neurochemical, and molecular mechanisms of alcohol dependence, with a major interest in the role of thecentralnucleusoftheamygdala(CeA) in excessive alcohol drinking. However, there is a great need to understand the mechanisms that mediate dependence- induced drinking motivated directly by withdrawal/abstinence symptoms. The Neurophysiology Project will continue to study the neuroadaptations induced by alcohol dependence in the CeA, and will now investigate upstream neuronal circuits that are responsible for dysregulation of the CeA during alcohol abstinence. Corticotropin-releasingfactor(CRF)andCRF1receptorsareinvolvedintheethanol-inducedincreaseinGABA releaseintheCeAandtheCRFsystemisupregulatedafterethanoldependence.ChronicCRF1antagonism blocked alcohol dependence-induced increases in ethanol consumption. Notably, the transition to alcohol dependencealsodysregulatesexecutivefunction,andtheinfralimbic(IL)subdivisionofthemPFCexerts?top- down?controlovertheamygdalatoregulateemotionalaspectsofgoal-directedbehaviors.Thus,thisrenewal application focuses on the overall hypothesis that ethanol dependence and withdrawal are driven by the recruitmentofCRFandserotonin(5-HT)signalingincortical-amygdalacircuits.Wewillstudyneuralfunction during ethanol abstinence and characterize how long those neuroadaptive changes persist. In particular, our goal is to characterize neuroadaptations in the CRF and 5-HT systems and their effects in the CeA and IL through common cellular systems to induce a maladaptation in neural function that promotes ethanol withdrawal-inducedanxiety-likebehavior.Ourprojectisdesignedtotestthehypothesisthatdependenceand withdrawal(earlywithdrawal=2-8h;?latewithdrawal=2weeks)differentiallyalterresponsesto5-HTandCRF inCeAneurons(SpecificAim1)andwillaffecttheexcitabilityofILpyramidalneuronsinlayerVthatprojectto the CeA (Specific Aim 2). Finally, Specific Aim 3 will electrophysiologically assess the ethanol-induced synaptic and molecular mechanisms of candidate drugs that are identified and tested in preclinical animal models (Contet and Animal Models Core) and subsequently tested in the clinical component (Mason). The project will use IL and CeA brain slices and standard whole-cell patch-clamp and cell-attached electrophysiologicalmethods,aswellasmeasuresofproteinlevelsandchemogeneticandbehavioraltesting that will involve continued collaboration with the George/Zorrilla, Contet, Martin-Fardon, and Mason projects andAnimalModelsCore.Itisimperativetoprovidedatathatelucidatethecellularbasisofthesusceptibilityof alcoholics to stress and relapse. A better understanding of the neuroadaptations that shape the synaptic networksthatareinvolvedinethanoldependencerepresentsachallengetoalcoholresearchersandwilllead tothedevelopmentofnewtherapeuticagentstoalleviatealcoholdependenceandpreventrelapse. PHS398/2590(Rev.06/09) Page ContinuationFormatPage